The major goal of the proposed research continues to be elucidation of the mechanisms involved in controlling the switch from latency to viral replication in Epstein-Barr virus (EBV) infected B lymphocytes. More specifically, this proposal focuses on the regulation of two linked EBV genes, the BZLF1 and BRLF1 genes, which are integrally involved in this switch. A central question with respect to maintenance of latency is whether the BZLF1 or BRLF1 promoters are actively repressed, or whether they merely require an 'activation" signal for promoter activity. During the previous funding period significant progress has been made identifying cis-elements within the BZLF1 promoter (Zp) involved in induction by TPA and anti-immunoglobulin. These studies also identified two cis-elements (ZIIIA and ZIIlB) in Zp involved in autoactivation by the BZLF1 gene product, Zta. In order to fully understand regulation of viral latency and induction of the lytic cycle it is critical to further characterize regulation of the BZLF1 and BRLF1 genes, and to identify and characterize those cellular factors which modulate their activities. Our proposed approach is as follows: a. Further characterization of the functional domains regulating Zp and Rp; (1) characterization of anti-Ig and butyrate inducibility of Zp and Rp; (2) functional analysis of the ZI domains in Zp; (3) characterization of cell and viral factor binding to the region from -129 to -105bp of Zp, which encompasses the Zta binding sites; (4) functional analyses of the ZII (AP1/CREB/C-EBF) domain in Zp; and (5) mapping cis-elements in Rp involved in induction; b. Generation of recombinant EBV harboring specific mutations in Zp or the BZLF1 gene; (1) incorporation of mutant Zp-reporters in the viral genome outside the BRLF1/BZLF1 locus; (2) generation of recombinant EBV with mutations within the BRLF1/BZLF1 locus; 2. Characterization and cloning of cellular transcription factors involved in regulating Zp and Rp activation; (l) analysis of cellular factors binding to functional domains in Zp and Rp; and (2) cloning cellular transcription factors involved in regulating expression of Zta and Rta; d. Investigation of the basis for variable inducibility of the lytic cycle in latently infected cell lines; (1) correlation of lytic cycle inducibility with inducibility of Zta and Rta expression; and (2) examination of cellular factors binding to cis-elements involved in regulating Zp and Rp in semipermissive versus nonpermissive cell lines.